The sodium/potassium-ATPase, or Na+ pump, is the protein responsible for the ATP dependent transport of sodium and potassium ions across cell membranes. While all cells of the body contain this essential enzyme, it is especially important in nerve and muscle because the electrical properties of the membrane are dependent on these two ion gradients. The transmembrane Na+ gradient maintained by the ATPase also provides the energy for numerous membrane co-transport mechanisms. The cardiac ATPase is the generally accepted receptor for digitalis, a drug commonly used in the treatment of congestive heart failure. Recent data indicate that a family of genes is responsible for different isoforms of the ATPase alpha subunit in species as diverse as chickens, rodents, and man. Only one isoform of the beta subunit has been identified in any species. The alpha subunit genes show tissue-specific expression, suggesting that the various ATPase isoforms have evolved to satisfy the ion transport needs of specific cells. The biological significance of ATPase isoforms will be examined by comparing functional differences between isoforms and by localizing the various isoforms to specific cells within complex tissues. Mouse L-cells will be transfected with chicken ATPase cDNA and the expression of the chicken subunits monitored by species-specific fluorescent antibody binding. Differences in ouabain sensitivity between the endogenous and expressed ATPase will allow pharmacological separation of mouse and chicken ATPase function. This expression system will be used to quantitate the ouabain sensitivity, ion transport activity, and ATPase activity of the expressed chicken isoforms. Co-expression of the alpha subunit isoforms with and without the beta subunit may identify an effect of the beta subunit on alpha subunit function. Isoform localization will be approached using two methods. In situ hybridization with isoform-specific cRNA probes will localize isoform mRNA to specific cells within brain, muscle, heart, and kidney. Isoform-specific antibodies will be produced against synthetic peptides and immunofluorescence techniques used to examine cell specific isoform expression. These site-directed antibodies will also be valuable reagents with which to study structure function relationships in future experiments. Correlation of ATPase isoform function with cellular localization will greatly increase our knowledge of ion transport physiology. Correlation of isoform function with the amino acid sequence substitutions that define each isoform will improve our understanding of the structure/function relationships involved in ion transport.